Abstract
Reverse transcription (RT) followed by polymerase chain reaction (RT-PCR) has been commonly used to detect viral and cellular transcripts in whole cell extracts. Application of this technique to tissue sections requires the in situ generation of cDNA. In this study, we selected an abundant transcript, Epstein-Barr virus (EBV)-encoded small RNA (EBER-1), as a model template to demonstrate cDNA generation in tissue sections. Using both digoxigenin-dUTP and primers which are complementary to EBER-1, we demonstrated specific EBER-1 cDNA generation both in vitro, and in tissue sections taken from formalin-fixed paraffin-embedded cell blocks of an EBV-infected cell line, B95-8. Furthermore, we utilized in situ RT in sections of EBV-associated nasopharyngeal carcinomas, and identified EBER-1 cDNA specifically in neoplastic cells, but not in the surrounding nonneoplastic stroma. EBER-1 cDNA was localized to the nucleus of these cells, with relative sparing of the nucleolus and the cytoplasm. No specific signal was evident if the reverse transcriptase was omitted, if ‘sense’ primers were used, or if RT was preceded by RNase digestion. The specificity of EBER-1 cDNA was further confirmed by in situ hybridization using the sense riboprobe, which has the same polarity as the EBER-1 transcript. Our results provide a successful example of using nonradioactive nucleotide analogue for cDNA generation in formalin-fixed, paraffin-embedded tissue sections. This approach would provide a visible assay to monitor RT in tissue sections, and allow further optimization of conditions for cDNA generation in tissue sections. Therefore, it potentially can be helpful for the future development of RT-PCR in tissue sections.
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References
Bagasra O, Hauptman SP, Lischner HW, Sachs M, Pomerantz RJ. Detection of human immunodeficiency virus type 1 provirus in mononuclear cells by in situ polymerase chain reaction. N Engl J Med 326:1385–1389;1992.
Bagasra O, Seshamma T, Pomerantz RJ. Polymerase chain reaction in situ intracellular amplification and detection of HIV-1 proviral DNA and other specific genes. J Immunol Methods 158:131–145;1993.
De Bault LE, Wang BL. Localization of mRNA by in situ transcription and immunogold-silver staining. Cell Vision 1:67–73;1994.
Embretson J, Zupancic M, Beneke J, Till M, Wolinsky S, Ribas JL, Burke A, Haase AT. Analysis of human immunodeficiency virus-infected tissues by amplification and in situ hybridization reveals latent and permissive infections at single-cell resolution. Proc Natl Acad Sci USA 90:357–361;1993.
Embretson J, Zupancic M, Ribas JL, Burke A, Racz P, Tenner-Racz K, Haase AT. Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS. Nature 362:359–362;1993.
Glickman JN, Howe JG, Steitz JA. Structural analysis of EBER-1 and EBER-2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol 62:902–911;1988.
Gressens P, Martin JR. In situ polymerase chain reaction: Localization of HSV-2 DNA sequences in infections of the nervous system. J Virol Methods 46:61–83;1994.
Gu J. Principles and applications of in situ PCR. Cell Vision 1:8–19;1994.
Haase AT, Retzel EF, Staskus KA. Amplification and detection of lentiviral DNA inside cells. Proc Natl Acad Sci USA 87:4971–4975;1990.
Heniford BW, Shum SA, Leonberger M, Hendler FJ. Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. Nucleic Acids Res 21:3159–3166;1993.
Howe JG, Steitz JA. Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization. Proc Natl Acad Sci USA 83:9006–9010;1986.
Isaacson SH, Asher DM, Gibbs CJ, Gajdusek DC. In situ RT-PCR amplification in archival brain tissue. Cell Vision 1:85;1994.
Komminoth P, Long AA, Ray R, Wolfe HJ. In situ polymerase chain reaction detection of viral DNA, single-copy genes, and gene rearrangements in cell suspensions and cytospins. Diagn Mol Pathol 1:85–97;1992.
Lerner MR, Andrews NC, Miller G, Steitz JA. Two small RNAs encoded by Epstein-Barr virus and complexed with protein are precipitated by antibodies from patients with systemic lupus erythematosus. Proc Natl Acad Sci USA 78:805–809;1981.
Long AA, Komminoth P, Lee E, Wolfe HJ. Comparison of indirect and direct in-situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 99:151–162;1993.
Nuovo GJ, MacConnell P, Forde A, Delvenne P. Detection of human papillomavirus DNA in formalin-fixed tissues by in situ hybridization after amplification by PCR. Am J Pathol 139:847–854;1991.
Nuovo GJ, Gallery F, MacConnell P, Becker J, Bloch W. An improved technique for the situ detection of DNA after polymerase chain reaction amplification. Am J Pathol 139:1239–1244;1991.
Nuovo GJ, Margiotta M, MacConnell P, Becker J. Rapid in situ detection of PCR-amplified HIV-1 DNA. Diagn Mol Pathol 1:98–102;1992.
Nuovo GJ, Gorgone GA, MacConnell P, Margiotta M, Gorevic PD. In situ localization of PCR-amplified human and viral cDNAs. PCR Methods Appl 2:117–123;1992.
Nuovo GJ, Lidonnici K, MacConnell P, Lane B. Intracellular localization of polymerase chain reaction-amplified hepatitis C cDNA. Am J Surg Pathol 17:683–690;1993.
Ray R, Komminoth P, Machado M, Wolfe HJ. Combined polymerase chain reaction and in situ hybridization for the detection of single-copy genes and viral genomic sequences in intact cells. Mod Pathol 4:124A;1991.
Soldo L, Mangano G, Milanese C. Synthesis of digoxigenin-labeled cDNA as a test for mRNA integrity. Biotechniques 13:876–879;1992.
Sallstrom JF, Zehbe I, Alemi M, Wilander E. Pitfalls of in situ polymerase chain reaction using direct incorporation of labelled nucleotides. Anticancer Res 13:1153;1993.
Staskus KA, Couch L, Bitterman P, Retzel EF, Zupancic M, List J, Haase AT. In situ amplification of visna virus DNA in tissue sections reveals a reservoir of latently infected cells. Microb Pathog 11:67–76;1991.
Tecott LH, Barchas JD, Eberwine JH. In situ transcription: specific synthesis of complementary DNA in fixed tissue sections. Science 240:1661–1664;1988.
Wappenschmidt C, Hausberg P, Jacobsen HJ. Synthesis of single-stranded digoxigenin-labeled cDNA and its use in differential hybridization. Colloquium. Boehringer Mannheim No 2, 6–7; 1990.
Wu TC, Mann RB, Charache P, Haywood SD, Staal S, Lambe BC, Ambinder RF. Detection of EBV gene expression in Reed-Sternberg cells of Hodgkin's disease. Int J Cancer 46:801–804;1990.
Wu TC, Mann RB, Epstein J, MacMahon E, Lee WA, Charache P, Hayward GS, Ambinder RF. Abundant expression of EBER-1 small nuclear RNA in nasopharyngeal carcinoma: A morphologically distinctive target detection of Epstein-Barr virus in formalin-fixed paraffin-embedded carcinoma specimens. Am J Pathol 138:1461–1469;1991.
Zehbe I, Hacker GW, Rylander E, Sallstrom J, Wilander E. Detection of single HPV copies in SiHa cells by in situ pooymerase chain reaction combined with immunoperoxidase and immunogold-silver staining techniques. Anticancer Res 12:2165–2168;1992.
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Wu, TC., Ling, Y., Kanayama, M.D. et al. Localization of epstein-barr virus-encoded small RNA-1 by in situ reverse transcription: Demonstration of cDNA generation in formalin-fixed paraffin-embedded tissue sections. J Biomed Sci 2, 249–255 (1995). https://doi.org/10.1007/BF02253385
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DOI: https://doi.org/10.1007/BF02253385